Enterically coated cysteamine, cystamine and derivatives thereof

ABSTRACT

The disclosure provides oral cysteamine and cystamine formulations useful for treating cystinosis and neurodegenerative diseases and disorders. The formulations provide controlled release compositions that improve quality of life and reduced side-effects.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/336,405, filed Oct. 27, 2016, which is a continuation of U.S.application Ser. No. 14/950,234, filed Nov. 24, 2015, which is acontinuation of U.S. application Ser. No. 14/752,383, filed Jun. 26,2015 (now U.S. Pat. No. 9,198,882), which is a divisional of U.S.application Ser. No. 14/555,993, filed Nov. 28, 2014, which is acontinuation of U.S. application Ser. No. 13/399,900, filed Feb. 17,2012, which is a continuation of U.S. application Ser. No. 13/190,396,filed Jul. 25, 2011 (now U.S. Pat. No. 8,129,433), which is a divisionalof U.S. application Ser. No. 11/990,869, filed Nov. 13, 2008 (now U.S.Pat. No. 8,026,284), which is a U.S. National Stage Application filedunder 35 U.S.C. § 371 and claims priority to International ApplicationNo. PCT/US07/02325, filed Jan. 26, 2007, which application claimspriority under 35 U.S.C. § 119 to U.S. Provisional Application Ser. No.60/762,715, filed Jan. 27, 2006, the disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to methods, compositions and treatments formetabolic conditions and free radical damage. More specifically, theinvention relates to methods and composition useful for treatingCystinosis and neurodegenerative diseases such as Huntington's,Alzheimer's and Parkinson's disease, as free radical andradioprotectants, and as hepto-protectant agents.

BACKGROUND

Cystinosis is a rare, autosomal recessive disease caused byintra-lysosomal accumulation of the amino acid cystine within varioustissues, including the spleen, liver, lymph nodes, kidney, bone marrow,and eyes. Nephropathic cystinosis is associated with kidney failure thatnecessitates kidney transplantation. To date, the only specifictreatment for nephropathic cystinosis is the sulfhydryl agent,cysteamine. Cysteamine has been shown to lower intracellular cystinelevels, thereby reducing the rate of progression of kidney failure inchildren.

Cysteamine, through a mechanism of increased gastrin and gastric acidproduction, is ulcerogenic. When administered orally to children withcystinosis, cysteamine has also been shown to cause a 3-fold increase ingastric acid production and a 50% rise of serum gastrin levels. As aconsequence, subjects that use cysteamine suffer gastrointestinal (GI)symptoms and are often unable to take cysteamine regularly or at fulldose.

To achieve sustained reduction of leukocyte cystine levels, patients arenormally required to take oral cysteamine every 6 hours, whichinvariably means having to awaken from sleep. However, when a singledose of cysteamine was administered intravenously the leukocyte cystinelevel remained suppressed for more than 24 hours, possibly becauseplasma cysteamine concentrations were higher and achieved more rapidlythan when the drug is administered orally. Regular intravenousadministration of cysteamine would not be practical. Accordingly, thereis a need for formulations and delivery methods that would result inhigher plasma, and thus intracellular, concentration as well as decreasethe number of daily doses and therefore improve the quality of life forpatients.

SUMMARY

The invention provides a composition comprising an enterically coatedcystamine or cystamine derivative.

The invention also provides a composition comprising an entericallycoated cysteamine or cysteamine derivative.

The invention further provides a composition comprising a coatedcystinosis therapeutic agent that has increased uptake in the smallintestine compared to a non-coated cystinosis therapeutic agent whenadministered orally. In one aspect, the coated cystinosis therapeuticagent comprises a cysteamine or cysteamine derivative.

The invention also provides a method of treating a subject withcystinosis, comprising administering to the subject a composition of theinvention.

The invention also contemplates a method of treating a subject with aneurodegenerative disease or disorder comprising administering to thesubject a composition of the invention comprising an enterically coatedcystamine or cystamine derivative.

The invention provides a pharmaceutical formulation comprising acomposition of the invention further including various pharmaceuticallyacceptable agents (e.g., flavorants, binders and the like) in apharmaceutically acceptable carrier.

The invention provides a method of treating cystinosis or aneurodegenerative disease or disorder comprising administering acomposition of the invention and a second therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-B shows enterocolonic tube. (A) Is an abdominal X-ray filmshowing the radiopaque weighted tip of the tube entering the ascendingcolon. (B) Is a contrast infused picture. The tube has passed throughthe small intestine and the tip is confirmed.

FIG. 2 shows mean plasma cysteamine levels taken from patients withcystinosis and control subjects after delivery of drug into variousintestinal sites. Error bars are standard error of the mean. In 2control subjects, most distal point of drug delivery was the mid-ilealregion.

FIG. 3 shows the mean change in leukocyte cystine levels, compared withbaseline levels, over a 12-hour period following delivery of cysteamineinto varying intestinal sites. Negative levels signify increasedleukocyte cystine depletion compared with baseline.

FIG. 4 shows a scatterplot of plasma cysteamine C_(max) vs. AOC of WBCCystine changes from Baseline. Positive value means decrease frombaseline. Negative value means increase from baseline. AOC change frombaseline was affected by C_(max) for cysteamine (P<0.001).

FIG. 5 shows serial leukocyte cystine levels after drug was given asnormal Cystagon® and enteric-coated (EC) cysteamine on alternate days.These serial levels were taken during the inpatient phase of the study.Desired cystine levels are below 1 mmol ½cystine/mg protein. Higher doseenteric-coated (yellow)) drug resulted in prolonged cystine suppressionwith 12 hour levels still within desired range.

FIG. 6 shows the blood cysteamine levels following a single 450 mg doseof Cystagon® (series 1), 450 mg EC-cysteamine (series 2) and 900 mgEC-cysteamine (series 3). The C_(max) is higher following EC drug. Inaddition, the time to C_(max) is longer following EC-drug, suggestingthat the drug is released from the capsule within the small intestinerather than the stomach.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a derivative” includes aplurality of such derivatives and reference to “a subject” includesreference to one or more subjects known to those skilled in the art, andso forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Cystinosis is a metabolic disease characterized by an abnormalaccumulation of the amino acid cystine in various organs of the bodysuch as the kidney, eye, muscle, pancreas, and brain. Different organsare affected at different ages.

There are three clinical forms of cystinosis. Infantile (ornephropathic) cystinosis; late-onset cystinosis; and benign cystinosis.The latter form does not produce kidney damage. Infantile cystinosis isusually diagnosed between 6 and 18 months of age with symptoms ofexcessive thirst and urination, failure to thrive, rickets, and episodesof dehydration. These findings are caused by a disorder called renaltubulopathy or Fanconi syndrome. As a consequence important nutrientsand minerals are lost in the urine. Children with cystinosis also havecrystals in their eyes (after one year of age) which may lead tophotosensitivity. They also have an increased level of cystine in theirwhite blood cells without adverse effect but allowing the diagnosis tobe ascertained. Without specific treatment, children with cystinosisdevelop end-stage renal failure, i.e., lose their kidney function,usually between 6 and 12 years of age. Without cysteamine treatmentsubjects can develop complications in other organs due to the continuedaccumulation of cystine throughout the body. These complications caninclude muscle wasting, difficulty swallowing, diabetes, andhypothyroidism.

Some symptoms include the inability of the kidneys to concentrate urineand allow important quantities of sodium, potassium, phosphorus,bicarbonate and substances like carnitine to be excreted in the urine.Treatment of symptoms compensates for these urinary losses. Subjectsneed to drink large quantities of water, because up to 2 to 3 liters ofwater are lost in the urine every day driving the feeling of beingthirsty. In addition, the loss of urinary electrolytes (sodium,potassium, bicarbonate, phosphorus) must be compensated in the subject.It is often necessary to add a salt supplement in the form of sodiumchloride. Children also lose bicarbonate and potassium in the urine,which can be compensated for by giving sodium bicarbonate and potassiumbicarbonate.

Specific treatments of cystinosis aim to reduce cystine accumulationwithin the cells. Cystinosis is currently treated with cysteamine(Cystagon®). Cysteamine also improves growth of cystinosis children.Cysteamine is only active in a very short period of time not exceeding5-6 hours, thus requiring administration of Cystagon® capsules 4 times aday, that is to say about every 6 hours. This treatment is also onlyeffective if continued day after day, indefinitely in order to controlthe disease. About 1000 children require lifelong treatment to prolongtheir lives and prevent deterioration of kidney function. However, asmentioned above, cysteamine administration results in increased gastricsecretions and is ulcerogenic. In addition, routes and timing ofadministration provide difficulty for subjects in need of such therapy.Recently, a similar drug called cystamine (the disulfide form ofcysteamine) has been studied for neurodegenerative disorders includingHuntington's and Parkinson's diseases. Cystamine has similarside-effects and dosing difficulties to that of cysteamine.

Cysteamine is a potent gastric acid-secretagogue that has been used inlaboratory animals to induce duodenal ulceration; studies in humans andanimals have shown that cysteamine-induced gastric acid hypersecretionis most likely mediated through hypergastrinemia. In previous studiesperformed in children with cystinosis who suffered regular uppergastrointestinal symptoms, a single oral dose of cysteamine (11-23mg/kg) was shown to cause hypergastrinemia and a 2- to 3-fold rise ingastric acid-hypersecretion. Symptoms suffered by these individualsincluded abdominal pain, heartburn, nausea, vomiting, and anorexia. Thedisclosure demonstrates that cysteamine-induced hypergastrinemia arises,in part, as a local effect on the gastric antral-predominant G-cells insusceptible individuals. The data also suggest that this is also asystemic effect of gastrin release by cysteamine. Depending upon theroute of administration, plasma gastrin levels usually peak afterintragastric delivery within 30 minutes, whereas the plasma cysteaminelevels peak later.

Subjects with cystinosis are required to ingest oral cysteamine(Cystagon®) every 6 hours, day and night. When taken regularly,cysteamine can deplete intracellular cystine by up to 90% (as measuredin circulating white blood cells), and this has been shown to reduce therate of progression to kidney failure/transplantation and also toobviate the need for thyroid replacement therapy. Unfortunately, becauseof the strict treatment regimen and the associated symptoms,non-adherence with cysteamine therapy remains a problem, particularlyamong adolescent and young adult patients. By reducing the frequency ofrequired cysteamine dosing, adherence to a therapeutic regimen can beimproved. The disclosure demonstrates that delivery of cysteamine to thesmall intestine reduces gastric distress and ulceration and improvesbioavailability of cysteamine in the circulation. Delivery of cysteamineinto the small intestine is useful due to improved absorption rate fromthe SI, greater surface area of the SI, and/or less cysteamineundergoing hepatic first pass elimination when absorbed through thesmall intestine. This disclosure shows a dramatic decrease in leukocytecystine within an hour of cysteamine delivery.

In addition, sulfhydryl (SH) compounds such as cysteamine, cystamine,and glutathione are among the most important and active intracellularantioxidants. Cysteamine protects animals against bone marrow andgastrointestinal radiation syndromes. The rationale for the importanceof SH compounds is further supported by observations in mitotic cells.These are the most sensitive to radiation injury in terms of cellreproductive death and are noted to have the lowest level of SHcompounds. Conversely, S-phase cells, which are the most resistant toradiation injury using the same criteria, have demonstrated the highestlevels of inherent SH compounds. In addition, when mitotic cells weretreated with cysteamine, they became very resistant to radiation. It hasalso been noted that cysteamine may directly protect cells againstinduced mutations. The protection is thought to result from scavengingof free radicals, either directly or via release of protein-bound GSH.An enzyme that liberates cysteamine from coenzyme A has been reported inavian liver and hog kidney. Recently, studies have appeareddemonstrating a protective effect of cysteamine against the hepatotoxicagents: acetaminophen, bromobenzene, and phalloidin.

Cystamine, in addition, to its role as a radioprotectant, has been foundto alleviate tremors and prolong life in mice with the gene mutation forHuntington's disease (HD). The drug may work by increasing the activityof proteins that protect nerve cells, or neurons, from degeneration.Cystamine appears to inactivate an enzyme called transglutaminase andthus results in a reduction of huntingtin protein (Nature Medicine 8,143-149, 2002). In addition, cystamine was found to increase the levelsof certain neuroprotective proteins. However, due to the current methodsand formulation of delivery of cystamine, degradation and poor uptakerequire excessive dosing.

The disclosure is not limited with respect to a specific cysteamine orcystamine salt or ester or derivative; the compositions of thedisclosure can contain any cysteamine or cystamine, cysteamine orcystamine derivative, or combination of cysteamine or cystamines. Theactive agents in the composition, i.e., cysteamine or cystamine, may beadministered in the form of a pharmacologically acceptable salt, ester,amide, prodrug or analog or as a combination thereof. Salts, esters,amides, prodrugs and analogs of the active agents may be prepared usingstandard procedures known to those skilled in the art of syntheticorganic chemistry and described, for example, by J. March, “AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure,” 4th Ed. (NewYork: Wiley-Interscience, 1992). For example, basic addition salts areprepared from the neutral drug using conventional means, involvingreaction of one or more of the active agent's free hydroxyl groups witha suitable base. Generally, the neutral form of the drug is dissolved ina polar organic solvent such as methanol or ethanol and the base isadded thereto. The resulting salt either precipitates or may be broughtout of solution by addition of a less polar solvent. Suitable bases forforming basic addition salts include, but are not limited to, inorganicbases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Preparation of estersinvolves functionalization of hydroxyl groups which may be presentwithin the molecular structure of the drug. The esters are typicallyacyl-substituted derivatives of free alcohol groups, i.e., moietieswhich are derived from carboxylic acids of the formula R—COOH where R isalkyl, and typically is lower alkyl. Esters can be reconverted to thefree acids, if desired, by using conventional hydrogenolysis orhydrolysis procedures. Preparation of amides and prodrugs can be carriedout in an analogous manner. Other derivatives and analogs of the activeagents may be prepared using standard techniques known to those skilledin the art of synthetic organic chemistry, or may be deduced byreference to the pertinent literature.

The disclosure provides delivery methods and compositions that overcomethe problems associated with cysteamine and cystamine delivery. Themethods of compositions of the disclosure provide enteric-coatedcompositions that result in less frequent dosing (2×/day vs. 4×/day),increased patient compliance and fewer gastrointestinal side effects(e.g., pain, heartburn, acid production, vomiting) and other sideeffects (e.g., patients smell like rotten eggs—a particular complianceproblem as subjects reach puberty). The disclosure providesenteric-coated cysteamine compositions (sulfhydryl/Cystagon®) andcystamine compositions.

The disclosure provides methods for the treatment of cystinosis, thetreatment of neurodegenerative disease such as Alzheimer Disease,Huntington's and Parkinson's disease and free radical damage usingenterically coated cysteamine and cystamine, respectively.

The disclosure provides composition comprising enterically formulatedcysteamine and cystamine derivatives. Examples of cysteamine derivativesinclude hydrochloride, bitartrate and phosphocysteamine derivatives.Cystamine and cystamine derivatives include sulfated cystamine. Entericcoatings prolong release until the cystamine, cystamine derivative, orcysteamine derivative/Cystagon® reaches the intestinal tract, typicallythe small intestine. Because of the enteric coatings, delivery to thesmall intestine is improved thereby improving uptake of activeingredient while reducing gastric side effects. This will result in areduction in the need for frequent administration that currently isassociated with Cystagon® therapy, cystamine and cysteamine therapy.

An “enterically coated” drug or tablet refers to a drug or tablet thatis coated with a substance—i.e., with an “enteric coating”—that remainsintact in the stomach but dissolves and releases the drug once the smallintestine is reached.

As used herein “enteric coating”, is a material, a polymer material ormaterials which encase the medicament core (e.g., cystamine, cysteamine,Cystagon®). Typically, a substantial amount or all of the entericcoating material is dissolved before the medicament or therapeuticallyactive agent is released from the dosage form, so as to achieve delayeddissolution of the medicament core. A suitable pH-sensitive polymer isone which will dissolve in intestinal juices at a higher pH level (pHgreater than 4.5), such as within the small intestine and thereforepermit release of the pharmacologically active substance in the regionsof the small intestine and not in the upper portion of the GI tract,such as the stomach.

The coating material is selected such that the therapeutically activeagent will be released when the dosage form reaches the small intestineor a region in which the pH is greater than pH 4.5. The coating may be apH-sensitive materials, which remain intact in the lower pH environs ofthe stomach, but which disintegrate or dissolve at the pH commonly foundin the small intestine of the patient. For example, the enteric coatingmaterial begins to dissolve in an aqueous solution at pH between about4.5 to about 5.5. For example, pH-sensitive materials will not undergosignificant dissolution until the dosage form has emptied from thestomach. The pH of the small intestine gradually increases from about4.5 to about 6.5 in the duodenal bulb to about 7.2 in the distalportions of the small intestine (ileum). In order to provide predictabledissolution corresponding to the small intestine transit time of about 3hours (e.g., 2-3 hours) and permit reproducible release therein, thecoating should begin to dissolve within the pH range of the duodenum,and continue to dissolve at the pH range within the small intestine.Therefore, the amount of enteric polymer coating should be sufficient tosubstantially dissolve during the approximate three hour transit timewithin the small intestine (e.g., the proximal and mid-small intestine).

Enteric coatings have been used for many years to arrest the release ofthe drug from orally ingestible dosage forms. Depending upon thecomposition and/or thickness, the enteric coatings are resistant tostomach acid for required periods of time before they begin todisintegrate and permit release of the drug in the lower stomach orupper part of the small intestines. Examples of some enteric coatingsare disclosed in U.S. Pat. No. 5,225,202 which is incorporated byreference fully herein. As set forth in U.S. Pat. No. 5,225,202, someexamples of coating previously employed are beeswax and glycerylmonostearate; beeswax, shellac and cellulose; and cetyl alcohol, masticand shellac, as well as shellac and stearic acid (U.S. Pat. No.2,809,918); polyvinyl acetate and ethyl cellulose (U.S. Pat. No.3,835,221); and neutral copolymer of polymethacrylic acid esters(Eudragit L30D) (F. W. Goodhart et al., Pharm. Tech., pp. 64-71, April1984); copolymers of methacrylic acid and methacrylic acid methylester(Eudragits), or a neutral copolymer of polymethacrylic acid esterscontaining metallic stearates (Mehta et al., U.S. Pat. Nos. 4,728,512and 4,794,001). Such coatings comprise mixtures of fats and fatty acids,shellac and shellac derivatives and the cellulose acid phthlates, e.g.,those having a free carboxyl content. See, Remington's at page 1590, andZeitova et al. (U.S. Pat. No. 4,432,966), for descriptions of suitableenteric coating compositions. Accordingly, increased adsorption in thesmall intestine due to enteric coatings of cystamine, cysteaminederivatives (including Cystagon®) can result in improvements incystinosis as well as neurodegenerative diseases including, for example,Huntington's disease.

Generally, the enteric coating comprises a polymeric material thatprevents cysteamine or cystamine release in the low pH environment ofthe stomach but that ionizes at a slightly higher pH, typically a pH of4 or 5, and thus dissolves sufficiently in the small intestines togradually release the active agent therein. Accordingly, among the mosteffective enteric coating materials are polyacids having a pK_(a) in therange of about 3 to 5. Suitable enteric coating materials include, butare not limited to, polymerized gelatin, shellac, methacrylic acidcopolymer type C NF, cellulose butyrate phthalate, cellulose hydrogenphthalate, cellulose proprionate phthalate, polyvinyl acetate phthalate(PVAP), cellulose acetate phthalate (CAP), cellulose acetatetrimellitate (CAT), hydroxypropyl methylcellulose phthalate,hydroxypropyl methylcellulose acetate, dioxypropyl methylcellulosesuccinate, carboxymethyl ethylcellulose (CMEC), hydroxypropylmethylcellulose acetate succinate (HPMCAS), and acrylic acid polymersand copolymers, typically formed from methyl acrylate, ethyl acrylate,methyl methacrylate and/or ethyl methacrylate with copolymers of acrylicand methacrylic acid esters (Eudragit NE, Eudragit RL, Eudragit RS). Forexample, the enterically coating can comprise Eudragit L30D,triethylcitrate, and hydroxypropylmethylcellulose (HPMC), Cystagon® (orother cysteamine derivative), wherein the coating comprises 10 to 13% ofthe final product.

By “pharmaceutically acceptable carrier” or “pharmaceutically acceptablevehicle” are meant materials that are suitable for oral administrationand not biologically, or otherwise, undesirable, i.e., that may beadministered to a subject along with an active ingredient withoutcausing any undesirable biological effects or interacting in adeleterious manner with any of the other components of a pharmaceuticalcomposition in which it is contained.

Similarly, a “pharmaceutically acceptable” salt, ester or otherderivative of an active agent comprise, for example, salts, esters orother derivatives which are not biologically or otherwise undesirable.

“Stabilizing agents” refer to compounds that lower the rate at whichpharmaceutical degrades, particularly an oral pharmaceutical formulationunder environmental conditions of storage.

By the terms “effective amount” or “therapeutically effective amount” ofa enteric formulation of cysteamine or cystamine refers to a nontoxicbut sufficient amount of the agent to provide the desired therapeuticeffect. As will be pointed out below, the exact amount required willvary from subject to subject, depending on the age, weight, and generalcondition of the subject, the severity of the condition being treated,and the like. An appropriate “effective” amount in any individual casemay be determined by one of ordinary skill in the art using only routineexperimentation.

In one aspect of the disclosure there is provided a stabilizedpharmaceutical composition for administration of an cysteamine orcystamine, wherein the cysteamine or cystamine is enterically coated.

The cysteamine or cystamine is present in the composition in atherapeutically effective amount; typically, the composition is in unitdosage form. The amount of cysteamine or cystamine administered will, ofcourse, be dependent on the age, weight, and general condition of thesubject, the severity of the condition being treated, and the judgmentof the prescribing physician. Suitable therapeutic amounts will be knownto those skilled in the art and/or are described in the pertinentreference texts and literature. In one aspect, the dose is administeredtwice per day at about 0.5-1.0 g/m² (e.g., 0.7-0.8 g/m²) body surfacearea. Current non-enterically coated doses are about 1.35 g/m² bodysurface area and are administered 4-5 times per day.

The entericaly coated cysteamine or cystamine can comprise variousexcipients, as is well known in the pharmaceutical art, provided suchexcipients do not exhibit a destabilizing effect on any components inthe composition. Thus, excipients such as binders, bulking agents,diluents, disintegrants, lubricants, fillers, carriers, and the like canbe combined with the cysteamine or cystamine. For solid compositions,diluents are typically necessary to increase the bulk of a tablet sothat a practical size is provided for compression. Suitable diluentsinclude dicalcium phosphate, calcium sulfate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch and powdered sugar.Binders are used to impart cohesive qualities to a tablet formulation,and thus ensure that a tablet remains intact after compression. Suitablebinder materials include, but are not limited to, starch (including cornstarch and pregelatinized starch), gelatin, sugars (including sucrose,glucose, dextrose and lactose), polyethylene glycol, waxes, and naturaland synthetic gums, e.g., acacia sodium alginate, polyvinylpyrrolidone,cellulosic polymers (including hydroxypropyl cellulose, hydroxypropylmethylcellulose, methyl cellulose, hydroxyethyl cellulose, and thelike), and Veegum. Lubricants are used to facilitate tablet manufacture;examples of suitable lubricants include, for example, magnesiumstearate, calcium stearate, and stearic acid, and are typically presentat no more than approximately 1 weight percent relative to tabletweight. Disintegrants are used to facilitate tablet disintegration or“breakup” after administration, and are generally starches, clays,celluloses, algins, gums or crosslinked polymers. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and the like. If desired, flavoring, coloring and/or sweeteningagents may be added as well. Other optional components for incorporationinto an oral formulation herein include, but are not limited to,preservatives, suspending agents, thickening agents, and the like.Fillers include, for example, insoluble materials such as silicondioxide, titanium oxide, alumina, talc, kaolin, powdered cellulose,microcrystalline cellulose, and the like, as well as soluble materialssuch as mannitol, urea, sucrose, lactose, dextrose, sodium chloride,sorbitol, and the like.

A pharmaceutical composition may also comprise a stabilizing agent suchas hydroxypropyl methylcellulose or polyvinylpyrrolidone, as disclosedin U.S. Pat. No. 4,301,146. Other stabilizing agents include, but arenot limited to, cellulosic polymers such as hydroxypropyl cellulose,hydroxyethyl cellulose, methyl cellulose, ethyl cellulose, celluloseacetate, cellulose acetate phthalate, cellulose acetate trimellitate,hydroxypropyl methylcellulose phthalate, microcrystalline cellulose andcarboxymethylcellulose sodium; and vinyl polymers and copolymers such aspolyvinyl acetate, polyvinylacetate phthalate, vinylacetate crotonicacid copolymer, and ethylene-vinyl acetate copolymers. The stabilizingagent is present in an amount effective to provide the desiredstabilizing effect; generally, this means that the ratio of cysteamineor cystamine to the stabilizing agent is at least about 1:500 w/w, morecommonly about 1:99 w/w.

The tablets are manufactured by first enterically coating the cysteamineor cystamine. A method for forming tablets herein is by directcompression of the powders containing the enterically coated cysteamineor cystamine, optionally in combination with diluents, binders,lubricants, disintegrants, colorants, stabilizers or the like. As analternative to direct compression, compressed tablets can be preparedusing wet-granulation or dry-granulation processes. Tablets may also bemolded rather than compressed, starting with a moist material containinga suitable water-soluble lubricant.

In an alternative embodiment, the enterically coated cysteamine orcystamine are granulated and the granulation is compressed into a tabletor filled into a capsule. Capsule materials may be either hard or soft,and are typically sealed, such as with gelatin bands or the like.Tablets and capsules for oral use will generally include one or morecommonly used excipients as discussed herein.

For administration of the dosage form, i.e., the tablet or capsulecomprising the enterically coated cysteamine or cystamine, a totalweight in the range of approximately 100 mg to 1000 mg is used. Thedosage form is orally administered to a patient suffering from acondition for which an cysteamine or cystamine would typically beindicated, including, but not limited to, cystinosis andneurodegenerative diseases such as Huntington's, Alzheimer's andParkinson's disease.

The compositions of the disclosure can be used in combination with othertherapies useful for treating cystinosis and neurodegenerative diseasesand disorders. For example, indomethacin therapy (Indocid® or Endol®) isan anti-inflammatory used to treat rheumatoid arthritis and lumbago, butit can be used to reduce water and electrolyte urine loss. In childrenwith cystinosis, indomethacin reduces the urine volume and thereforeliquid consumption by about 30%, sometimes by half. In most cases thisis associated with an appetite improvement. Indomethacin treatment isgenerally followed for several years.

Other therapies can be combined with the methods and compositions of thedisclosure to treat diseases and disorders that are attributed or resultfrom cystinosis. Urinary phosphorus loss, for example, entails rickets,and it may be necessary to give a phosphorus supplement. Carnitine islost in the urine and blood levels are low. Carnitine allows fat to beused by the muscles to provide energy. Hormone supplementation issometimes necessary. Sometimes the thyroid gland will not produce enoughthyroid hormones. This is given as thyroxin (drops or tablets). Insulintreatment is sometimes necessary if diabetes appears, when the pancreasdoes not produce enough insulin. These treatments have become rarelynecessary in children whom are treated with cysteamine, since thetreatment protects the thyroid and the pancreas. Some adolescent boysrequire a testosterone treatment if puberty is late. Growth hormonetherapy may be indicated if growth is not sufficient despite a goodhydro electrolytes balance. Accordingly, such therapies can be combinedwith the enterically coated cysteamine and cystamine compositions andmethods of the disclosure.

The effectiveness of a method or composition of the disclosure can beassessed by measuring leukocyte cystine concentrations. Dosageadjustment and therapy can be made by a medical specialist dependingupon, for example, the severity of cystenosis and/or the concentrationof cystine. Additional therapies including the use of omeprazole(Prilosec®) can reduce these symptoms.

In addition, various prodrugs can be “activated” by use of theenterically coated cysteamine. Prodrugs are pharmacologically inert,they themselves do not work in the body, but once they have beenabsorbed, the prodrug decomposes. The prodrug approach has been usedsuccessfully in a number of therapeutic areas including antibiotics,antihistamines and ulcer treatments. The advantage of using prodrugs isthat the active agent is chemically camouflaged and no active agent isreleased until the drug has passed out of the gut and into the cells ofthe body. For example, a number of produgs use S—S bonds. Weak reducingagents, such as cysteamine, reduce these bonds and release the drug.Accordingly, the compositions of the disclosure are useful incombination with pro-drugs for timed release of the drug. In thisaspect, a pro-drug can be administered followed by administration of anenterically coated cysteamine compositions of the invention (at adesired time) to activate the pro-drug.

It is to be understood that while the invention has been described inconjunction with specific embodiments thereof, that the foregoingdescription as well as the examples which follow are intended toillustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention

Examples

Subjects.

Children with cystinosis, ≤12 years old, and taking regular cysteaminebitartrate (Cystagon®; Mylan, Morgantown, W. Va.) were recruited to thestudy (Table I). Adult control patients were recruited locally. Patientswith cystinosis had a mean leukocyte cystine level of less than 2.0 nmolhalf-cystine/mg protein over the past year. Cysteamine therapy wasdiscontinued 2 days before admission, and acid suppressants,antibiotics, nonsteroidal anti-inflammatory drugs, pro-kinetic agents,and antihistamines were discontinued 2 weeks before admission. None ofthe patients had undergone kidney transplantation. Baseline chemistry,Helicobacter pylori serologic study, complete blood count, andurinalysis were performed.

TABLE I Cystinosis patient data Serum Age Weight Cysteamine creatininePatient (yrs.) Sex (kg) dose (mg)* (mg/dL) 1 16 Male 61.5 500 1.0 2 14Male 39.4 406 1.2 3 13 Female 39.1 406 1.5 4 19 Female 38.1 406 1.4 5 13Female 50.1 500 1.0 6 16 Male 58.7 500 3.1 *Dose of cysteamine basedelivered into varying delivery sites

Cysteamine Bitartrate Delivery.

Cysteamine was infused through a silicone rubber nasoenteric tube(Dentsleeve Pty Ltd, Australia), 3 mm in diameter and 4.5 meters long.The tube, specifically made for this study, had a tungsten-weighted tip,and immediately proximal to this was an inflatable balloon (5-mLcapacity). Immediately proximal to the balloon was an infusion port (1mm diameter) through which the drug was delivered. After an overnightfast (except for water), the dose of cysteamine bitartrate (10mg/kg/dose of base, maximum of 500 mg) was dissolved in 10 mL of waterand infused over 1 to 2 minutes. On day 1 of the study, the nasoenterictube was inserted into the stomach. By day 3 of the study the tube hadpassed into the proximal small intestine (SI) just distal to theligament of Treitz (confirmed fluoroscopically). The balloon was theninflated, and peristalsis propelled the tube distally. Tube positionwithin the cecum was confirmed fluoroscopically on day 5 (day 7 in 4patients because of slow transit). If the tube had migrated too far, itwas retracted into the desired location.

Serum Gastrin, Cysteamine and Leukocyte Cystine Measurements.

After an overnight fast (except for water) blood samples were taken atbaseline and at varying intervals after intraluminal delivery ofcysteamine. Serum gastrin levels were then measured at 30, 60, 90, and120 minutes and 3 and 4 hours; cysteamine levels were measured at 0, 5,10, 20, 30, 45, 60, 75, 90, 105, 120, and 150 minutes and 3, 4, 6, 8,10, 12, and 16 hours; leukocyte cystine levels were measured at 1, 2, 3,4, 6, and 12 hours in patients with cystinosis only. Gastrin wasmeasured in picograms/mL with the Diagnostic Products Corporation (LosAngeles, Calif.) gastrin radioimmunoassay-assay kit. Leukocyte cystinelevels were measured in nmol half-cystine per mg protein by the CystineDetermination Lab (La Jolla, Calif.).

To measure plasma cysteamine, 100-μL plasma samples were collected inheparinized vacutainers and spun in a centrifuge within 1 hour, andplasma was stored at −18° C. The concentration of cysteamine wasmeasured by use of tandem mass spectroscopy (API 2000 LC/MS/MS; AppliedBiosystems, Foster City, Calif.). Cysteamine concentrations werecalculated with a calibration curve that was prepared by spiking plasmawith buffered cysteamine solutions, and quality control samples wereanalyzed with each batch.

Statistical Analysis.

Mixed model restricted maximum likelihood (REML) repeated measuresanalysis of variance with subjects as a random effect was performed onthe absolute leukocyte cystine levels, on the leukocyte cystine levelchanges from baseline, and on the “area over the curve” (AOC) forleukocyte cystine level changes from baseline after cysteamineadministration for the subjects with cystinosis. AOC is computationallyanalogous to area under the curve, but it is applied when values arepredominantly decreasing below baseline values. Large AOC values reflectlarge decreases, and a negative AOC reflects a net increase in value.Main effects for site of delivery, time after delivery, and theinteraction between site and time were tested, except just the siteeffect was tested for AOCs. In the absence of significant interactionwhen a main effect was detected, Tukey's honestly significant differencetest (HSD) was applied to identify where differences occurred within a5% family wise error rate. The Tukey HSD procedure controls for overallsignificance level when performing all pairwise comparisons. Anadditional analysis was performed with plasma cysteamine C_(max) addedto the AOC model.

REML repeated measures analyses of variance with subjects as a randomeffect were also performed as described above on AUC and the C_(max)over time for plasma cysteamine levels separately for the subjects withcystinosis and control subjects and with both subject groups combined.Differences between means for the 3 sites were tested, plus group andgroup x site interaction effects for the combined groups. If a siteeffect was detected, Tukey's HSD was applied to determine which sitesdiffered from each other.

REML repeated measures analyses of variance were also performed asdescribed above on gastrin levels. The analyses were performed on 2versions of datasets: the full dataset and all data after omittingobservations collected at 30 minutes (1 subject was missing a bloodsample taken at 30 minutes after small intestinal cysteamine delivery).A 5% significance level was used without adjustment for all statisticaltesting.

Six patients with cystinosis, (3 male, 3 female) with a mean age of 15.2years (range 13-19 years) were recruited into the study (Table I). Eighthealthy adult control patients (6 male, 2 female) with a mean age of23.2 years (range 19-28 years) were enrolled. None of the children withcystinosis had undergone kidney transplantation. All control subjectsreceived 500 mg cysteamine base, whereas the mean dose for subjects withcystinosis was 453 mg (range 406-500 mg). All subjects had normal liverfunction test results. In all subjects the nasoenteric tube passedsuccessfully from the stomach into the upper SI; however, it did notprogress any further in 2 subjects with cystinosis. In 2 of the controlsubjects the tube only reached the mid-ileum but did, however, progressto the cecum in 8 subjects (4 control subjects, 4 with cystinosis).There were no reported adverse effects with the insertion or removal ofthe nasoenteric tube (FIG. 1).

Symptoms.

Only 2 patients (1 male, 1 female) with cystinosis reported regular GIsymptoms before the study, and these had responded to acid-suppressiontherapy. The male subject had severe retching and emesis about 15minutes after receiving intragastric cysteamine but did not have anysymptoms when the drug was infused into the proximal small intestine.The female child with cystinosis had mild transient nausea after SI drugdelivery only. No other symptoms were reported after any othercysteamine delivery in the children with cystinosis. There were noassociated adverse events with tube placement or removal.

Plasma Cysteamine.

Among the subjects with cystinosis as measured by analysis of variance,the mean plasma cysteamine C_(max) and AUCs (of the concentration-timegradient) differed by site of cysteamine delivery (both P<0.03). Site(^(†)) refers to either patients with cystinosis or control subjects.For the plasma cysteamine AUCs, the means differed between the duodenaland both gastric and cecal sites of delivery (Tukey HSD global P<0.05).Among control subjects, the mean AUC did not differ among delivery sites(P>0.4), but mean C_(max) did (P<0.05). For both cystinosis and controlgroups the mean C_(max) values differed only between the duodenum andcecum; mean C_(max) values after duodenal versus gastric or gastricversus cecal delivery were not statistically different (Tables II andIII).

TABLE II Mean plasma cysteamine C_(max) levels (γmol/L) and area undercurve (AUC) measurements in cystinosis subjects, controls, and combinedcystinosis and control subjects, after delivery of cysteamine into thestomach, small intestine, and cecum C_(max) AUC C_(max) AUC C_(max) AUCCys- Cys- Con- Con- Com- Com- tinosis tinosis trol trol bined binedStomach 35.5 3006 39.5 3613 37.8 3353 (20.5) (1112) (16.4) (1384) (17.6)(1267) Small 55.8 4299 51.1 3988 53.2 4047 Intestine (13.0) (1056)(20.7) (1659) (17.4) (1376) Cecum 21.9 3002 23.1 2804 22.5 2903 (13.1) (909) (15.3) (1323) (13.2) (1056)The Standard Deviations are in Parenthesis

TABLE III Comparisons of mean plasma cysteamine C_(max) (γmol/L) and AUCmeasurements for combined cystinosis subjects and control subjects amongdelivery sites AUC C_(max) P value* <0.01 <0.01 Stomach vs SI + +Stomach vs Cecum − − SI vs Cecum + + + Significant difference usingTukey's HSD test (α = 0.05) − No significant difference *ANOVA test forequality of three delivery sites

When data from the control subjects were combined with cystinosissubject data, there was both a group effect (P<0.05) and a site effect(P<0.01) for AUCs, with a significant difference between mean AUC levelsfor the duodenum versus both the stomach and cecum. C_(max) valuesdiffered among sites (P<0.01) but not between groups (P>0.4). Group (*)refers to site of intestinal delivery. C_(max) differed between duodenumversus both stomach and cecum (FIG. 2).

Leukocyte Cystine.

There were significant differences among the 3 sites of delivery forcystine levels (P<0.04), changes from baseline values (P<0.0001), andAOCs for changes from baseline (P<0.02). A Tukey HSD test, whichcontrols for multiple comparisons, showed that mean leukocyte cystinelevels differed between the cecum and stomach sites, but that cecumversus duodenum and stomach versus duodenum produced similar meanvalues. When the absolute cystine levels or AOCs for changes frombaseline levels were evaluated, the significant differences in siteswere found between the duodenum and both the stomach and cecum, but notbetween stomach and cecum (Tukey HSD global P<0.05) (FIG. 3). Plasmacysteamine C_(max) and AUC contributed a statistical effect on AOC(P<0.001 and <0.02, respectively), even after controlling for deliverysite (FIG. 4).

Blood Gastrin.

For the full gastrin dataset, there was a significant difference amongthe means for the different delivery sites (P<0.1), with the cecumresulting in a lower mean from that of the stomach and small intestine.Both group * and site † significant effects were detected after omittingobservations from 30 minutes after delivery (P<0.05 and P<0.01,respectively). The 30-minute observations were omitted because of amissing data set. For these observations, mean levels of gastrin afterdelivery in the cecum were different from those from both the duodenumand stomach, although the latter did not differ from each other. The 1boy (14 years) who had severe GI symptoms after intragastric, but notenteric or cecal, cysteamine delivery had a rise in baseline gastrinfrom 70 pg/mL to 121 pg/mL at 30 minutes after gastric cysteamine.Within the control group, more than half of the baseline andpost-cysteamine gastrin levels remained undetectable (<25 pg/mL), andnone of the control subjects had a significant rise in gastrin aftercysteamine delivery into any site.

Patients with cystinosis are required to ingest oral cysteamine(Cystagon®) every 6 hours, day and night. When taken regularly,cysteamine can deplete intracellular cystine by up to 90% (as measuredin circulating white blood cells), and this has been shown to reduce therate of progression to kidney failure/transplantation and also toobviate the need for thyroid replacement therapy. Unfortunately, becauseof the strict treatment regimen and the associated symptoms,non-adherence with cysteamine therapy remains a problem, particularlyamong adolescent and young adult patients. Certainly, by reducing thefrequency of required cysteamine dosing adherence can be improved. Thedisclosure shows a strong statistical association between the maximumplasma concentration (C_(max)) of cysteamine and AOC measurements forleukocyte cystine (P<0.001). A higher C_(max) is achieved after deliveryof cysteamine into the small intestine than when infused into thestomach or colon; this may be due to improved absorption rate from theSI, greater surface area of the SI, or less cysteamine undergoinghepatic first pass elimination when absorbed rapidly through the smallintestine. When data were combined for patients with cystinosis andcontrol subjects, there was a statistical difference between duodenalversus both gastric and colonic delivery for plasma cysteamine C_(max)and AUC levels (both P<0.05). The lack of similar statisticalsignificance for the cystinosis group alone may simply reflect the smallnumber of patients studied. Changes from baseline leukocyte cystinelevels were statistically significant for absolute cystine levels andfor AOC when cysteamine was infused into the duodenum compared with bothstomach and colon. As shown in FIG. 3, the leukocyte cystine levelsremained below pre-delivery levels for up to 12 hours after a singledose of cysteamine into the small intestine. This would suggest thateffective absorption of cysteamine through the SI, by causing a higherC_(max) and AUC on the cysteamine concentration-time gradient, couldlead to prolonged depletion of leukocyte cystine and possibly lessfrequent daily dosing. Another explanation would be that by achieving ahigh enough plasma cysteamine concentration, more drug reaches thelysosome (where cystine accumulates). In the lysosome the cysteaminereacts with cystine forming the mixed disulfide of cysteamine andcysteine. The mixed disulfide exits the lysosome presumably via thelysine carrier. In the cytosol the mixed disulfide can be reduced by itsreaction with glutathione. The cysteine released can be used for proteinor glutathione synthesis. The cysteamine released from the mixeddisulfide reenters the lysosome where it can react with another cystinemolecule. Thus 1 molecule of cysteamine may release many molecules ofcystine from the lysosome. This study showed a dramatic decrease inleukocyte cystine within an hour of cysteamine delivery. In retrospect,the finding from this study was that the leukocyte cystine levelsremained at the 1-hour level for 24 hours, and even at 48 hours afterdelivery the levels had not returned to the pre-cysteamine level.

Cysteamine is a potent gastric acid-secretagogue that has been used inlaboratory animals to induce duodenal ulceration; studies in humans andanimals have shown that cysteamine-induced gastric acid hypersecretionis most likely mediated through hypergastrinemia. In previous studiesperformed in children with cystinosis who suffered regular uppergastrointestinal symptoms, a single oral dose of cysteamine (11-23mg/kg) was shown to cause hypergastrinemia and a 2- to 3-fold rise ingastric acid-hypersecretion. Symptoms suffered by these individualsincluded abdominal pain, heartburn, nausea, vomiting, and anorexia.Interestingly, only 2 of 6 subjects with cystinosis (who were known tosuffer regular cysteamine-induced GI symptoms) had increased gastrinlevels and symptoms, including nausea, retching, and discomfort afterintragastric cysteamine. Gastrin levels were only available after smallintestinal administration in 1 of the 2 children and the levels remainedthe same as baseline. Neither child had symptoms after entericcysteamine delivery. None of the other patients with cystinosis orcontrol subjects had an increase in gastrin levels with cysteamineinfused into any site. This would suggest that cysteamine-inducedhypergastrinemia may arise as a local effect on the gastricantral-predominant G-cells only in susceptible individuals. In addition,plasma gastrin levels usually peaks after intragastric delivery within30 minutes, whereas the plasma cysteamine levels peaked later. 8, 10 In2 previous studies, children with cystinosis were shown to have asignificant rise in plasma gastrin levels after receiving intragastriccysteamine; as part of these study's entry criteria all subjects did,however, suffer with regular GI symptoms. Data from this study wouldsuggest that cysteamine does not cause hypergastrinemia, and thereforeacid-hypersecretion, in all patients with cystinosis. Thus acidsuppression therapy would not be recommended in patients with cystinosiswithout upper GI symptoms.

The data suggest that direct administration of cysteamine into thejejunum may result in prolonged leukocyte cystine depletion. In aprevious study, a child who had a gastrojejunal feeding tube for oralfeeding aversion and severe UGI symptoms, responded to intrajejunalcysteamine with a 3-fold rise in serum gastrin as compared with drugadministration into the stomach. The leukocyte cystine response was notmeasured in this child. Therefore patients with jejunal feeding tubeswill have to be further evaluated.

FIGS. 5 and 6 shows results from a patient that remained on the twicedaily EC-cysteamine for an extended period of time. Over this period thepatient's leukocyte cystine levels have been measured regularly. Thedose of twice daily EC-cysteamine is titrated against the patient'ssymptoms and cystine levels. The patient's cystine levels have been 0.4,1.0, 0.36.

This study provides data that may be used to improve the quality of lifefor patients with cystinosis. The present formulation of Cystagon®comprises cysteamine in a capsule that will dissolve rapidly on contactwith water, most likely within the stomach.

Although a number of embodiments and features have been described above,it will be understood by those skilled in the art that modifications andvariations of the described embodiments and features may be made withoutdeparting from the teachings of the disclosure or the scope of theinvention as defined by the appended claims.

What is claimed is:
 1. A dosage form comprising enterically-coatedgranules having a core comprising cysteamine bitartrate and cystamine ora pharmaceutically-acceptable salt thereof; at least onepharmaceutically acceptable binder; and an enteric coating surroundingthe core, wherein the enterically-coated granules release cysteamine andcystamine or a pharmaceutically-acceptable salt thereof at a pH of4.5-6.5.
 2. A method of treating a subject with cystinosis comprisingadministering a composition of claim 1 less than 4 times per day,wherein the total daily dose of cystamine is about 1.35 g/m² bodysurface area or less.
 3. A method of treating a subject with cystinosiscomprising administering a composition of claim 1, wherein eachadministration comprises an amount of cysteamine bitartrate that wouldyield a dose of 100 to 1000 mg cysteamine free base.
 4. A method oftreating a subject with cystinosis comprising administering acomposition of claim 1 less than 4 times per day in an amount to providewhite blood-cell cystine suppression with a 12 hour level below 1nmol/½cystine/mg protein.
 5. A method of treating a subject withcystinosis comprising administering a composition of claim 1 twice perday in an amount to provide white blood-cell cystine suppression with a12 hour level below 1 nmol/½cystine/mg protein.
 6. A method of treatinga subject with Huntington's Disease comprising administering thecomposition of claim 1, wherein each administration comprises an amountof cysteamine bitartrate that would yield a dose of 100 to 1000 mgcysteamine free base.